Multigene phylogeny and morphology reveal Ophiocordyceps hydrangea sp. nov. and Ophiocordyceps bidoupensis sp. nov. (Ophiocordycipitaceae)

MycoKeys 92: | 09_| 30 (2022) er-reviewed open-access journal

doi: 10.3897/mycokeys.92.86 | 60 < Mycokeys

https://mycokeys.pensoft. net Launched to accelerate biodiversity research

Multigene phylogeny and morphology reveal
Ophiocordyceps hydrangea sp. nov. and Ophiocordyceps
bidoupensis sp. nov. (Ophiocordycipitaceae)

Weiqiu Zou! , Dexiang Tang!?,, Zhihong Xu', Ou Huang",
Yuanbing Wang', Ngoc-Lan Tran’, Hong Yu!

| Yunnan Herbal Laboratory, College of Ecology and Environmental Sciences, Yunnan University, Kunming
650504, Yunnan, China 2 School of Life Science, Yunnan University Kunming 650504, Yunnan, China
3 Institute of Regional Research and Development, Ministry of Science and Technology, Hanoi, Vietnam

Corresponding author: Hong Yu (hongyu@ynu.edu.cn, herbfish@163.com)

Academic editor: Cecile Gueidan | Received 5 May 2022 | Accepted 10 August 2022 | Published 30 August 2022

Citation: Zou W, Tang D, Xu Z, Huang O, Wang Y, Tran N-L, Yu H (2022) Multigene phylogeny and morphology
reveal Ophiocordyceps hydrangea sp. nov. and Ophiocordyceps bidoupensis sp. nov. (Ophiocordycipitaceae). MycoKeys 92:
109-130. https://doi.org/10.3897/mycokeys.92.86160

Abstract

Ophiocordyceps species have a wide range of insect hosts, from solitary beetle larva to social insects. However,
among the species of Ophiocordyceps, only a few attack cicada nymphs. These species are mainly clustered
in the Ophiocordyceps sobolifera clade in Ophiocordyceps. A new entomopathogenic fungus parasitic on
cicada nymphs, and another fungus parasitic on the larva of Coleoptera, are described in this study. The
two new species viz. Ophiocordyceps hydrangea and Ophiocordyceps bidoupensis were introduced based on
morphology and multigene phylogenetic evidence. The phylogenetic framework of Ophiocordyceps was re-
constructed using a multigene (nrSSU, nrLSU, tef-1a, rpb1, and rpb2) dataset. The phylogenetic analyses
results showed that O. hydrangea and O. bidoupensis were statistically well-supported in the O. sobolifera
clade, forming two separate subclades from other species of Ophiocordyceps. The distinctiveness of these

two new species was strongly supported by both molecular phylogeny and morphology.

Keywords

2 new taxa, entomopathogenic fungi, morphology, phylogenetic analyses

* Those authors contributed equally to this work.

Copyright Weigiu Zou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

110 Weigiu Zou et al. / MycoKeys 92: 109-130 (2022)

Introduction

Ophiocordyceps G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora is the largest genus in
the Ophiocordycipitaceae, comprising approximately 290 species. It was originally es-
tablished by Petch, with Ophiocordyceps blattae Petch as the type species (Petch 1931).
According to the arrangement of the perithecia, the size of asci, ascospores, and sec-
ondary ascospores, Ophiocordyceps was transferred to Cordyceps sensu lato by Kobayasi,
as a subgenus of Cordyceps s.l. (Kobayasi 1941, 1982). Sung et al. (2007) used five
to seven loci combined molecular datasets to revise the Cordyceps and the Clavicipi-
taceae. The species of Cordyceps and Clavicipitaceae were divided into three families
(Cordycipitaceae, Ophiocordycipitaceae, Clavicipitaceae sense stricto) and four genera
(Cordyceps sense stricto, Ophiocordyceps, Elaphocordyceps, and Metacordyceps). The re-
search results of Sung et al. (2007) are currently the most widely accepted phylogenetic
classification of Cordyceps s.1. In 2015, Ophiocordyceps was divided into O. ravenelii
clade, O. unilateralis clade, O. sobolifera clade, and O. sphecocephala clade by Sanjuan
et al. With the continuous revision of Ophiocordyceps, it has now been divided into four
clades, including the Hirsutella clade, O. sobolifera clade, O. sphecocephala clade, and
O. ravenelii clade (Mains 1958; Sung et al. 2007; Quandt et al. 2014; Sanjuan et al
2015; Simmons et al. 2015; Wang et al. 2018). Many phylogenetic classifications for
entomopathogenic fungi have been revised in recent studies (Wang et al. 2018; Fan et
al. 2021; Wang et al. 2021a, 2021b).

There are fewer species in the O. sobolifera clade than in the Hirsutella clade and
the O. sphecocephala clade. The O. sobolifera clade is statistically well-supported in most
studies and 11 species have been described in the Index Fungorum (Kobayasi and
Shimizu 1963; Hywel-Jones 1995b; Sung et al. 2007, 2011; Luangsa-ard et al. 2008;
Hyde et al. 2017; Crous et al. 2018, 2019; Lao et al. 2021; Wang et al. 2021a). Asexual
morphs of Ophiocordyceps were reported as Hirsutella Pat., Paraisaria Samson & B.L.
Brady, Sorosporella Sorokin, Hymenostilbe Petch and Syngliocladium Petch, etc. (Sung
et al. 2007; Quandt et al. 2014). In most species of Ophiocordyceps, their dominant
asexual morphs were Hirsutella, the conidiogenous cells basally swollen that taper to a
narrow neck, producing a mucilaginous cluster of one or several conidia (Simmons et
al. 2015; Wang et al. 2018).

Ophiocordyceps species have a wide range of insect hosts, from solitary bee-
tle larvae to social insects. More than 10 insect orders were attacked, including
Hemiptera, Coleoptera, Lepidoptera, Blattaria, Dermaptera, Diptera, Hymenop-
tera, Isoptera, Megaloptera, and Mantodea (Araujo et al. 2015; Araujo and Hughes
2016, 2019). Entomopathogenic fungi whose hosts are cicada nymphs have at-
tractive stromata. The most typical representative of this group was Cordyceps ci-
cadae (Miquel) Massee (Massee 1895) in Cordycipitaceae, with the stroma like
a flower (Sung et al. 2007). However, for species of Ophiocordyceps, with cicada
nymph hosts including O. khonkaenensis Tasanathai, Thanakitpipattana & Luang-
sa-ard (Crous et al. 2019), O. sobolifera (Hill ex Watson) G.H. Sung, J.M. Sung,

Multigene phylogeny and morphology of two new species 111

Hywel-Jones & Spatafora (Kobayasi and Shimizu 1963; Sung et al. 2007), and O.
longissima (Kobayasi) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora (Kobayasi
and Shimizu 1963; Sung et al. 2007, 2011) in O. sobolifera clade, their stromata
were typically bright-colored and cylindrical. The hosts of the entomopathogenic
fungi within the O. sobolifera clade were divided into two categories. One group
with Hemiptera hosts was represented by O. sobolifera. These fungi had a hard
texture stroma, which was cylindrical, and deep-colored, and had swollen fertile
parts (Kobayasi and Shimizu 1963; Sung et al. 2011; Crous et al. 2019). Another
group had Coleoptera hosts that were characterized by hard texture stromata, being
cylindrical, bright-colored, and with a sterile apices cone at the top of the stroma
(Hywel-Jones 1995b; Luangsa-ard et al. 2008; Crous et al. 2018; Lao et al. 2021;
Wang et al. 2021a).

Cordyceps s.l. is globally distributed with the highest species diversity recorded
in subtropical and tropical regions (Nguyen and Vo 2005; Ban et al. 2015; Doan et
al. 2017; Luangsa-ard et al. 2018), especially in East and Southeast Asia (Sung et al.
2007; Fan et al. 2021; Wang et al. 2021a). To date, more than 800 species of Cordyceps
and Ophiocordyceps have been named worldwide, and there are at least 200 species in
China (Index Fungorum 2022). Yunnan Province, located in southwest China, has
unique geographical and ecological features. Many species of Ophiocordyceps were re-
ported from Yunnan, including O. alboperitheciata H. Yu, Q. Fan & Y.B. Wang (Fan
et al. 2021), O. furcatosubulata H. Yu, Y. Wang & Y.B. Wang (Wang et al. 2021a),
O. highlandensis Zhu L. Yang & J. Qin (Yang et al. 2015), O. lanpingensis H. Yu
& Z.H. Chen (Chen et al. 2013), O. laojunshanensis J.Y. Chen, Y.Q. Cao & D.R.
Yang (Chen et al. 2011), O. langshanensis (M. Zang, D.Q. Liu & R.Y. Hu) H. Yu,
Y. Wang, Y.D. Dai, Zhu L. Yang & Y.B. Wang (Wang et al. 2021b), and O. pingbi-
anensis H. Yu, S.Q. Chen & Y.B. Wang (Chen et al. 2021). The unique geographical
conditions of Yunnan have resulted in high Cordyceps s.l. species diversity. There is
also a high species diversity of Cordyceps s.1. in Southeast Asia, where more than 500
species of entomopathogenic fungi have been reported. Approximately 400 species of
entomopathogenic fungi are distributed in Thailand (Sung et al. 2007; Luangsa-ard
et al. 2011, 2018; Ban et al. 2015; Tasanathai et al. 2019; Xiao et al. 2019). Vietnam
is second to Thailand, in the number of entomopathogenic fungi species, with more
than 100 species having been reported such as Moelleriella pumatensis T.T. Nguyen
& N.L. Tran (Mongkolsamrit et al. 2011), O. furcatosubulata H. Yu, Y. Wang & Y.B.
Wang (Wang et al. 2021a), and O. puluongensis H. Yu, Z.H. Xu, N.L. Tran & Y.B.
Wang (Xu et al. 2022). These findings suggested that Vietnam should be abundant
in species diversity of Cordyceps s.l. (Mongkolsamrit et al. 2011; Doan et al. 2017;
Luyen et al. 2017).

Several studies have evaluated the taxonomy and biology of entomopathogenic
fungi, especially species found in China and Southeast Asia. In this study, one un-
known species of Ophiocordyeps attacking a cicada nymph was collected from Yun-

nan Province, Jinghong City, Nabanhe National Nature Reserve, in China. Another

112

Weigiu Zou et al. / MycoKeys 92: 109-130 (2022)

Table |. Specimen information and GenBank accession numbers of the sequences used in this study.

Species

Hirsutella citriformis
Hirsutella fusiformis

Hirsutella gigantea
Hirsutella guyana
Hirsutella illustris
Hirsutella kirchneri
Hirsutella lecaniicola
Hirsutella liboensis

Hirsutella necatrix
Hirsutella nodulosa

Hirsutella radiata

Hirsutella rhossiliensis

Hirsutella strigosa
Hirsutella subulata

Hirsutella thompsonii vat.
synnematosa

Hirsutella thompsonii vat.
thompsonii

Hirsutella thompsonii vat.
vinacea

Ophiocordyceps acicularis
Ophiocordyceps acicularis
Ophiocordyceps agriotidis
Ophiocordyceps annulata
Ophiocordyceps aphodi

Ophiocordyceps appendiculata

Ophiocordyceps arborescens

Ophiocordyceps bidoupensis

Ophiocordyceps bidoupensis

Ophiocordyceps brunneanigra
Ophiocordyceps
brunneaperitheciata
Ophiocordyceps
brunneipunctata
Ophiocordyceps citrina
Ophiocordyceps cochlidiicola

Ophiocordyceps cossidarum

Ophiocordyceps crinalis
Ophiocordyceps evansii

Ophiocordyceps formicarum

Host

Cixiidae (Hemiptera)
Brachyderes incanus
(Curculionidae,
Coleoptera)
Pamphiliidae
(Hymenoptera)
Empoasca kraemeri
(Cicadellidae, Hemiptera)
Eriosoma lanigerum
(Aphididae, Hemiptera)
Abacarus hystrix
(Eriophyidae, Acari)
Parthenolecanium corni
(Coccidae, Hemiptera)
Larva of Cossidae
(Lepidoptera)
Acari
Dioryctria zimmermani
(Pyralidae, Lepidoptera)
Diptera
Mesocriconema xenoplax

(Criconematidae,

Tylenchida)
Nephotettix virescens
(Cicadellidae, Hemiptera)
Microlepidoptae
(Lepidoptera)
Aceria sheldoni
(Eriophyidae, Acari)
Phyllocoptruta oleivora
(Eriophyidae, Acari)
Acalitus vaccinii
(Eriophyidae, Acari)
Larva of Coleoptera
Larva of Coleoptera
Larva of Coleoptera
Larva of Coleoptera
Larva of Scarabaeidae
(Coleoptera)
Larva of Coleoptera
Larva of Pueraria lobata
(Lepidoptera)
Larva of Elateridae
(Coleoptera)
Larva of Elateridae
(Coleoptera)
Cicadellidae (Hemiptera)
Larva of Lepidoptera

Larva of Elateridae
(Coleoptera)
Hemiptera
Cochlididae pupa
(Lepidoptera)
Larva of Cossidae
(Lepidoptera)
Larva of Lepidoptera
Pachycondyla harpax adult
ant (Hymenoptera)
Formicidae
(Hymenoptera)

Isolate no./
specimen no.

ARSEF 1446
ARSEF 5474
ARSEF 30
ARSEF 878
ARSEF 5539
ARSEF 5551
ARSEF 8888
ARSEF 9603

ARSEF 5549
ARSEF 5473

ARSEF 1369
ARSEF 3747
ARSEF 2197
ARSEF 2227
ARSEF 2459
ARSEF 137
ARSEF 254

OSC 110987
OSC 110988
ARSEF 5692
CEM 303
ARSEF 5498

NBRC 106960
NBRC 105891

YFCC 8793
YHH 20036

TBRC 8093
TBRC 8100

OSC 128576

TNS F18537
HMAS 199612

MEPLU 17-0752

GDGM 17327
HUA 186159

TNS F18565

nrSSU
KM652065
KM652067

KMG652068
KM652069
KMG652070
KM652071
KM652072

KM652073
KM652074

KM652076
KM652080
KM652085
KM652086
KM652099
KM652087
KM652101

EF468950
EF468951
DQ522540
KJ878915
DQ522541

JN941728
AB968386

OM304638

OK571396

DQ522542

KJ878917
MF398186

KF226253
KC610796

KJ878921

GenBank accession no.

orLSU

KM652106
KM652110
JX566977

KM652111
KM652112
KM652113
KM652114
KM652115

KM652116
KM652117

KM652119
KM652123
KM652129
KM652130
KM652147
KM652131
KM652149

EF468805
EF468804
DQ518754
KJ878881
DQ518755

JN941413
AB968414

MF614654
MF614658

DQ518756

KJ878903
KJ878884

MF398187

KF226254
KC610770

KJ878888

tef-1u

KM651990
KM651993
JX566980

KM651994
KM651996
KM651997
KM651998
KY415588

KM651999
KM652000

KM652002
KM652006
KM652012
KM652013
KM652027
KM652014
KM652028

EF468744
EF468745
DQ522322
KJ878962
DQ522323

AB968577
AB968572

OK556894
OK556893

MF614638
MF614643

DQ522324

KJ878983
KJ878965

MF928403

KF226256
KC610736

KJ878968

rpbl rpb2
KM652031 -
KM652033 =
KM652034 -

KM652035 =

KM652037 =

KMG652038 -
KY945367 =

KM652039 -
KM652040 -

KM652042 -
KM652045 -
KM652050 -
KM652051 -
KM652061 -
KM652052 -
KM652062 -

EF468852 =
EF468853 =
DQ522368 DQ522418
KJ878995 -

L DQ522419

JN992462 AB968539

- AB968534
OK556898 OK556900
OK556897 OK556899

MFG614668 MF614681
= MEG14685
DQ522369 DQ522420

e KJ878954
KJ878998 ©

MF928404 -

KF226255 a
KP212916 =

KJ879002 KJ878946

Multigene phylogeny and morphology of two new species

Species Host
Ophiocordyceps forquignonii Adult fly (Diptera)
Ophiocordyceps Larva of Elateridae
Jurcatosubulata (Coleoptera)
Ophiocordyceps Larva of Elateridae
Jurcatosubulata (Coleoptera)
Ophiocordyceps geometridicola Larva of Geometridae

(Lepidoptera)
Ophiocordyceps Larva of Coleoptera
houaynhangensis
Ophiocordyceps hydrangea Nymph of cicada
(Hemiptera)
Ophiocordyceps hydrangea Nymph of cicada
(Hemiptera)
Ophiocordyceps hydrangea Nymph of cicada
(Hemiptera)
Ophiocordyceps Rarstii Hepialus jianchuanensis
(Lepidoptera)

Ophiocordyceps kimflemingiae Camponotus castaneus/
americanus (Hymenoptera)

Ophiocordyceps kniphofioides — Cephalotes atratus adult ant

(Hymenoptera)
Ophiocordyceps konnoana Larva of Coleoptera
Ophiocordyceps langbianensis Larva of Coleoptera
Ophiocordyceps lanpingensis Larva of Hepialidae
(Lepidoptera)
Ophiocordyceps longissima Cicada nymph (Cicadidae,
Hemiptera)
Ophiocordyceps longissima Hemiptera; cicada
(nymph)
Ophiocordyceps Larva of Cossidae
macroacicularis (Lepidoptera)
Ophiocordyceps Lepidoptera larva
multiperitheciata
Ophiocordyceps myrmicarum Hymenoptera
(Formicidae)
Ophiocordyceps nigrella Larva of Lepidoptera
Ophiocordyceps pruinosa Hemiptera
Ophiocordyceps Larva of Lepidoptera
pseudoacicularis
Ophiocordyceps pulvinata Camponotus adult ant
(Hymenoptera)
Ophiocordyceps ramosissimum Phassus nodus larva
(Lepidoptera)
Ophiocordyceps ravenelii Beetle larva (Coleoptera)
Ophiocordyceps robertsii Larva of Hepialidae
(Lepidoptera)
Ophiocordyceps Larva of Coleoptera
rubiginosiperitheciata
Ophiocordyceps satoi Polyrhachis lamellidens
(Hymenoptera)
Ophiocordyceps sinensis Larva of Hepialidae
(Lepidoptera)
Ophiocordyceps sinensis Larva of Hepialidae
(Lepidoptera)
Ophiocordyceps sobolifera Cicada nymph (Cicadidae,
Hemiptera)
Ophiocordyceps sobolifera Hemiptera (cicada
nymph)
Ophiocordyceps spataforae Hemiptera adult

Ophiocordyceps sphecocephala Hymenoptera adult wasp
Larva of Elateridae
(Coleoptera)

Hymenotera adult ant

Ophiocordyceps stylophora

Ophiocordyceps thanathonensis

Isolate no./
specimen no.

OSC 151902
YFCC 904

YHH 17005
TBRC 8095
TBRC 8428
YFCC 8832
YECC 8833
YECC 8834

MFLU:15-3884

SCO9B
HUA 186148
EFCC 7315
DL0017
YHOS0705

NBRC 106965
EFCC 6814

NBRC 100685
BCC 69008

HIRS 45
EFCC 9247
NHJ 12994
TBRC 8102

TNS-F 30044

GZUHHN8

OSC 110995
KEW 27083

NBRC 106966
y9
EFCC 7287
YHH 1805
TNS F18521
NBRC 106967
NHJ 12525
NBRC 101753

OSC 110999

MELU 16-2910

nrSSU
KJ878912
MT1774216

MT1774217

OM304636
OM304637
OM304635
KU854952
KX713631
KC610790

EF468959
MT928355
KC417458

AB968392

AB968388

KJ680150

EF468963
EU369106

GU904208
kJ028012

DQ522550

JN941704
KX713650
EF468971
MK984568
KJ878933
AB968395

EF469125
JN941695
EF468982

MF882926

GenBank accession no.

orLSU
KJ878876
MT774223

M1774224
MF614648
MH092902
OM304640
OM304641

OM304639

KX713620
KF658679
MT928306
KC417460
AB968420
EF468817

AB968416
MF614657
JX566965

EF468818
EU369041
MF614646

AB721305

DQ518764
EF468826

JN941437
KX713601
EF468827
MK984580
KJ878898
AB968422

EF469078
JN941446
EF468837

MF850377

tef-1u.

MT774244
MT774245
MF614632
MH092894
OM831277
OM831278
OM831276
KU854945

KX713698

KC610739

EF468753

KC417462
AB968584
EF468757
AB968574
MF614641
JX566973

EF468758
EU369024
MF614630

GU904209
kJo28014

DQ522334
EF468766

AB968582
KX713684
EF468767
MK984572
KJ878979
AB968590

EF469063
AB968592
EF468777

MF872614

rpbl
kJ878991
M1774230
M1774231

MF614663

OM831280
OM831281
OM831279
KU854943
KX713724
KF658667

EF468861

KC417464

EF468865

KJ680151

EF468866
EU369063
MF614661

GU904210
KJ028017

DQ522379

JN992438
KX713710
EF468874
MK984587

KJ879013

EF469092
JN992429
EF468882

MF872616

Ths

rpb2
KJ878945
M1774237
MT1774238

MF614679

OM831283
OM831284

OM831282

KC610717

EF468916

KC456333

AB968546

AB968536

MF614682

EF468920
EU369084
MF614677

DQ522430

AB968544

EF468924

MK984576

EF469111
AB968553
EF468931

114 Weiqiu Zou et al. / MycoKeys 92: 109-130 (2022)

Species Host Isolate no./ GenBank accession no.
specimen no. nrSSU nt LSU tef-1e. rpb1 rpb2
Ophiocordyceps tiputinii Larva of Megaloptera QCNE 186287 KC610792 KC610773 KC610745 KF658671 -
Ophiocordyceps tricentri Adult of Cercopoidea NBRC 106968 —_AB968393 AB968423 AB968593 - AB968554
(Hemiptera)

Ophiocordyceps unilateralis Camponotus sericeiventris VIC 44303 KX713628 KX713626 KX713675 KX713730 -

s. st. (Hymenoptera)

Ophiocordyceps unituberculata Larva of Lepidoptera YFCC HU1301 KY923214 KY923212 KY923216 KY923218 KY923220

Ophiocordyceps xuefengensis Larva of Phassus nodus GZUH2012HN14 KC631789 - KC631793 KC631798 -
(Lepidoptera)

Ophiocordyceps yakusimensis Cicada nymph (Cicadidae, HMAS 199604 — kjJ878938 —kJ878902 - kJ879018 KJ878953
Hemiptera)

Paraisaria amazonica Adult of Acrididae HUA 186143 KJ917562.—-KJ917571 =KM411989 KP212902 KM411982
(Orthoptera)

Paraisaria coenomyiae Coenomyia sp. NBRC 106964 AB968385 AB9G68413 AB968571 - AB968533

(Coenomyiidae, Diptera)

Paraisaria gracilis Larva of Lepidoptera EFCC 8572 EF468956 EF468811 EF468751 EF468859 EF468912

Paraisaria heteropoda Cicada nymph NBRC 100644 JN941718 JN941423 AB968596 JN992452 AB968557
(Hemiptera)

Tolypocladium inflatum Coleoptera (larva) OSC 71235 EF469124 EF469077 EF469061 EF469090 EF469108

Tolypocladium ophioglossoides Fungi (Elaphomyces sp.) CBS 100239 KJ878910 KJ878874 KJ878958 KJ878990 _‘KJ878944

unknown species of Ophiocordyeps attacking larvae of Elateridae was collected from
Lintong Province, Bidoup Nuiba National Park, in Vietnam. The phylogeny and mor-
phology of these two fungi were determined, and their systematic position was estab-
lished in Ophiocordycipitaceae. The phylogenetic analyses results showed that the two
new species belonged to Ophiocordyceps, and were named Ophiocordyceps hydrangea and
Ophiocordyceps bidoupensis based on well-supported morphology and molecular data.

Materials and methods

Sample collection and isolation

The specimens were collected from China and Vietnam, and the collection site
information was noted, including altitude, longitude, latitude, and habitat type.
Samples were placed in sterilized tubes or plastic bags and boxes, returned to the
laboratory, and stored at 4 °C. The specimens were photographed using a Canon 750
D camera (Canon Inc., Tokyo, Japan). The size was measured, and characteristics
were recorded including length of the stroma, single or multiple, length and width
of stipe clavate and fertile parts, shape, texture, and color. To obtain axenic cultures,
the segments were removed from insect bodies, and these segments were placed
onto Potato Dextrose Agar (PDA) consisting of peptone and yeast powder (potato
100 g/500 mL, dextrose 10 g/500 mL, agar 10 g/500 mL, yeast powder 5 g/500 mL,
peptone 2.5 g/500 mL) plates. The plates were placed in a culture room at 25 °C
until isolated into pure cultures. The cultures were saved on a PDA slant (to grow
slowly), and stored at 4 °C. All specimens were deposited in the Yunnan Herbal
Herbarium (YHH) of Yunnan University. The extypes of the two species were
deposited in the Yunnan Fungal Culture Collection (YFCC) of Yunnan University.

Multigene phylogeny and morphology of two new species LS

Morphological observations

To describe the sexual morphs of the two species, frozen sections or hand sec-
tions of the fruiting structures of the stroma were immersed in water and then
dyed with lactophenol cotton blue solution for morphological observation and
photomicrography (Wang et al. 2021a). For observations on asexual morphs, new
colonies were established from old cultures and placed on new PDA plates. The
plates were cultured in an incubator for 6 or 12 weeks at 25 °C, and then asexual
morphs were observed and recorded (shape, texture, and color of the colonies).
Microscope slide cultures were made using the methods of Wang et al. (2020). The
morphological observations and measurements were made using Olympus CX40

and BX53 microscopes.

DNA extraction, PCR, and sequencing

Five-centimeter segments from the stroma of fresh specimens and the cultures were
used for DNA extraction to ensure the cultures and specimens were the same. Total
DNA was extracted using cetyltrimethyl ammonium bromide (CTAB) according to
the procedure described by Liu et al. (2001). The DNA was used for PCR ampli-
fication. The primer pair, NS4 (5'-CTTCCGTCAATTCCTTTAAG-3') and NS1
(5'-GTAGTCATATGCTTGTCTC-3') was used to amplify nrSSU (the nuclear ribo-
somal small subunit) (White et al. 1990). The primer pair, LR5 (5'-ATCCTGAGG-
GAAACTTC-3') and LROR (5'-GTACCCGCTGAACTTAAGC-3') was used to
amplify nrLSU (the nuclear ribosomal large subunit) (Vilgalys and Hester 1990; Reh-
ner and Samuels 1994). The primer pair, 983F (5'-GCYCCYGGHCAYCGTGAY-
TTYAT-3') and 2218R (5'-ATGACACCRACRGCRACRGTYTG-3') was used to
amplify zef-J« (the translation elongation factor 1x) (Rehner and Buckley 2005). The
primer pair, CRPB1A (5'-CAYCCWGGYTTYATCAAGAA-3') and RPBIC (5'-CC-
NGCDATNTCRITRICCATRIA-3') were used to amplify 7p4/ (the largest subunit
of RNA polymerase II) (Castlebury et al. 2004; Bischoff et al. 2006). The primer
pair, FRPB2-5F (5'-GAYGAYMGWGATCAYTTYGG-3’) and fRPB2-7cR (5'-CCC-
ATRGCTTGYTTRCCCAT-3') was used to amplify 7p62 (the second largest subunit
of RNA polymerase II) (Liu et al. 1999). The polymerase chain reaction (PCR) for am-
plification of the five genes and their sequencing were described by Wang et al. (2015).

Phylogenetic analyses

Sequences of the five genes (nrSSU, nrLSU, tef-1a, rpb1, and rpb2) were downloaded
from GenBank, and combined with the newly generated sequences in this study. The
taxa information of the species and GenBank accession numbers of the five genes are
listed in Table1. Sequences of the five genes were aligned using the Clustal X (v.2.0) and
MEGAG (v.6.0) (Larkin et al. 2007; Tamura et al. 2013). Ambiguously aligned sites
were eliminated, and the gaps were treated as missing data. The aligned sequences of
the five genes (nrSSU, nrLSU, tef-1a, rpb1, and rpb2) were concatenated into a single

116 Weigiu Zou et al. / MycoKeys 92: 109-130 (2022)

combined dataset using MEGAG (v.6.0.). Conflicts between the five genes were tested
using PAUP* (v.4.0b10) (Swofford 2002). The results of the phylogenetic signals in the
five genes were not in conflict. The concatenated dataset containing all five genes con-
sisted of 11 data partitions, including one each for nrSSU and nrLSU, and three for each
of the three codon positions of tef-1a, rpb1, and rpb2. Phylogenetic analyses based on
the five genes were made using BI and ML methods (Ronquist and Huelsenbeck 2003;
Stamatakis et al. 2008). We used the optimal model GTR+I with 1,000 rapid boot-
strap replicates on the five genes for ML analyses (Stamatakis 2006). We conducted BI
analyses using a GIR+G+I model determined by jModelTest (v.2.1.4), conducted on
MrBayes (v.3.1.2) for 5 million generations (Darriba et al. 2012). The phylogenetic tree
constructed was viewed and edited using Fig Iree (v.1.4.2) and Adobe Illustrator CS6.

Results

Phylogenetic analyses

A total of 83 samples were used for the phylogenetic analyses. Five gene sequences of
the two new species collected were used to reconstruct the phylogenetic framework
of Ophiocordyceps. Two taxa of Tolypocladium were designated as the outgroup, and
these were, respectively, Tolypocladium ophioglossoides CBS 100239 and Tolypocladium
inflatum OSC 71235. The alignment lengths of the 83 samples were composed of
4,486 bp sequence data, 971 bp of nrSSU, 921 bp of nrLSU, 943 bp of tef-1a, 726 bp
of rpb1, and 925 of rpb2. The phylogenetic tree showed that these were identical in
overall topologies to previous studies. Four clades (Hirsutella clade, O. sobolifera clade,
O. sphecocephala clade, and O. ravenelii clade) of Ophiocordyceps were well-supported
by ML bootstrap proportions and BI posterior probabilities (Fig. 1). The two new
species in the O. sobolifera clade, O. hydrangea and O. bidoupensis, formed two sepa-
rate subclades. Three samples of O. hydrangea (BP = 98%, PP = 1) formed a separate
subclade with O. longissima and O. yakusimensis, while O. bidoupensis (BP = 83%,
PP = 0.99) formed a separate subclade with O. houaynhangensis.

Taxonomy

Ophiocordyceps hydrangea H. Yu, W.Q. Zou & D.X. Tang, sp. nov.
MycoBank No: 843203
Fig. 2

Etymology. Hydrangea, referred to the top of the stroma similar to hydrangea.
Holotype. Curna, Yunnan Province, Jinghong City, Nabanhe National Nature

Reserve, 22°8'21.32"N, 100°42'18.35"E, alt. 612 m, on cicada nymphs (Cicadidae,

Hemiptera). The material was found in the soil of an evergreen broad-leaved forest, 18

August 2020, H. Yu (YHH 20081, holotype; YFCC 8834, ex-holotype culture).

Multigene phylogeny and morphology of two new species ie

i100 Ophiocordycep
0.81/8 Ophiocordyceps si
0.93/57 Ophiocordyceps
0.99/22Mleme Ophiocordyceps robe

0.63/83 Ophiocordyceps :
et | Ophiocordyceps lanp!
BINA Ophiocordyceps mi

Hirsutella page
i100 OPhiocor

1/68| Hirsutella.
SHE q Ophiocordyceps

0.97/62
iT Ophiocordyceps app

Ophiocordyceps si
oof Ophiocordyce
Ophiocordyceps aci
i100 Ophiocordyceps arborescens
Ophiocordyceps ramosis.
Hirsutella rhossiliensis A
Hirsutella lecaniicolc
Ophiocordyceps.
Hirsutella kirchneri ARSEF 5551
Ophiocordyceps crinalis GDGM 17327
Ophiocordyceps brunneanigra TBRC 8093
Ophiocordyceps spataforae NHJ 12525
Hirsutella guyana ARSEF 878
phic east pruinosa Buu 12994

0.78/35 saat

0.65/30 7

0.99/43

0.61/59

1/100} 0.99/99] Ophiocordyceps pseudoa
0ONL Hirsutella nodulosa ARSEF 5473
onan Hirsutella subulata ARSEF 2227
Ophiocordyceps brunneaperitheciata TBRC 8100
Ophiocordyceps agriotidis ARSEF 5692
Hirsutella necatrix ARSEF 5549
ON oo 10of/Zirsutella thompsonii var. vinacea ARSEF 254
1/1008 Hirsutella thompsonii var. thompsonii ARSEF 137
Hirsutella thompsonii var. synnematosa ARSEF 2459
a Ophiocordyceps pulvinata TNS-F 30044
Ophiocordyceps satoi J19
0.99/84 aa. .
‘ial 1/100 Ophiocordyceps unilateralis VIC 44303 Hirsutella ant
Ophiocordyceps kimflemingiae SC09B pathogen subclade
Ophiocordyceps tiputinii QCNE 186287
Ophiocordyceps kniphofioides HUA 186148
Hirsutella citriformis ARSEF 1446 at ris
Hirsutella gigantea ARSEF 30
woop Hirsutella radiata ARSEF 1369
Hirsutella fusiformis ARSEF 5474
Hirsutella liboensis ARSEF 9603 =
Ophiocordyceps myrmicarum HIRS 45
af OPHiocordyceps ;
0.99/87 = Ophiocordyceps h
1/98 Ophiocordyceps hyd
Be |_| Ophiocordyceps longissima
a 100 al Ophiocordyceps longiss:
aa | | Ophiocordyceps yakus
L_ Ophiocordyceps sobolifer
Ophiocordyceps sobolife
1/100} Ophiocordyceps

0.99/83]

H. thompsonii subclade

0.57/12 1/93

0.60/53

0.96/44.

1/100} —

Ophiocordyceps konnoana EFCC 7
1/1001 Ophiocordyceps ravenelii OSC 1
Mb] _ Lophiocordveps nigra BCC 924
Paraisaria gracilis EFCC 8572
Paraisaria amazonica HUA 186143 |

Paraisaria
PT ee NBRC 100644 |

100 Tol) pease inflatum OSC Toa
Tolypocladium ophioglossoides CBS 100239

0.02 —_

Figure |. Phylogenetic relationships of Ophiocordyceps hydrangea and related species from the five genes
dataset (nrLSU, nrSSU, tef-1a, rpb1, and rpb2) based on ML and BI analyses. Statistical support values of
BI posterior probabilities and ML bootstrap proportions (0.5/250%) are shown at the nodes.

118 Weigiu Zou et al. / MycoKeys 92: 109-130 (2022)

Sexual morph. The stroma was grown from the head of the host cicada nymph,
solitary, the top of the stroma similar to hydrangea, pale pink, 1.6—6.4 cm long. Sexual
morph was not observed.

Asexual morph. ‘The colony grew slowly on PDA medium. Cultured at 25 °C
for about 12 weeks, the diameter of the colony was 25-28 mm, pale pink, the edge
white, hard texture. The back of the colony was white to brown. Surface hyphae rough,

Figure 2. Ophiocordyceps hydrangea A, B fungus on a cicada nymph C, D colony on PDA medium

E conidiophores, conidiogenous cells and conidia F-J conidiogenous cells and conidia K conidia. Scale

bars: 1 cm (A, B); 2 cm (C, D); 10 um (E, FG, I, J); 5 pm (H, K).

Multigene phylogeny and morphology of two new species 119

hyaline, septate. Conidiophores were cylindrical. Conidiogenous cells were solitary or
whorled, ampuliform, smooth-walled, forming on conidiophores or colonies, hyaline,
with swollen base, and slender top, 10.6—17.6 um long, 2.9—4.3 um wide at the swol-
len base, and 1.1—2.2 um wide at the slender top. Conidia hyaline, ovoid or long oval,
solitary, 6.8-10.1 x 3.3-4.5 um.

Host. Cicada nymph (Cicadidae, Hemiptera).

Habitat. In the soil of an evergreen broad-leaved forest.

Distribution. China.

Other material examined. Cuina, Yunnan Province, Jinghong City, Nabanhe
National Nature Reserve, 22°8'21.32"N, 100°42'18.35"E, alt. 612 m, on cicada
nymphs (Cicadidae, Hemiptera) was found in the soil an evergreen broad-leaved for-
est, 18 August 2020, H. Yu (YFCC 8832, YFCC 8833).

Notes. Phylogenetic analyses showed that O. hydrangea clustered with O. sobolif-
era, O. longissima, and O. yakusimensis of the O. sobolifera clade (Fig. 1). Their hosts
were cicada nymphs compared to other species of the O. sobolifera clade (Table 2).
Ophiocordyceps hydrangea was well supported by BI and ML results, forming a separate
subclade with O. sobolifera, O. longissima, and O. yakusimensis. The macro-morphology
of O. hydrangea was clearly different from O. sobolifera, O. longissima, O. khonkaenen-
sis, and O. yakusimensis. The stroma of O. hydrangea grew from the head of the host
cicada nymph, solitary, and the top of the stroma was like a pale pink hydrangea.

Ophiocordyceps bidoupensis H. Yu, W.Q. Zou & D.X. Tang; sp. nov.
MycoBank No: 843204
Fig. 3

Etymology. Bidoupensis, referred to the type species collected from Bidoup Nuiba
National Park.

Holotype. VietNam, Lintong Province, Bidoup Nuiba National Park, 12°8'9.30"N,
108°31'51.38"E, alt. 1678 m, on larva of Elateridae (Coleoptera) buried in soil, emerg-
ing from the leaf litter on the forest floor, 16 October 2017, H. Yu (YHH 20036, holo-
type; YFCC 8793, ex-holotype culture).

Sexual morph. The stroma grew from the head of the host, solitary, solid, cylin-
drical, 11.8—22.5 cm long, yellow. Stipe clavate, yellow, curved, 10.7—21.2 cm long,
0.7-0.9 mm wide. Fertile parts cylindrical, yellow, slightly curved, 2.9-11.3 mm long,
0.9-1.6 mm wide. Sterile apices cone, yellow, 2.1—-7.2 mm long, 0.2-0.7 mm wide.
Perithecia immersed, pyriform to lanceolate, brown-yellow, 213.4-405.9 x 74.8-
192.4 um. Asci hyaline, slender, 116.1-192.7 x 4.8-7.5 um. Asci cap prominent,
capitate, 4.7—6.1 x 3.3-5.4 um. Ascospores hyaline, filiform, multi-septate.

Asexual morph. The colony grew slowly on PDA medium. Cultured at 25 °C
for about 6 weeks, the diameter of the colony was 38-45 mm, white, aerial myce-
lium on the surface, slightly convex. The back of the colony was grayish-white, dark
brown in the middle. Surface smooth of hyphae, hyaline, septate. Conidiogenous cells
cone, hyaline, septate, smooth-walled, forming on hyphae, with a hypertrophic base,

120 Weigiu Zou et al. / MycoKeys 92: 109-130 (2022)

tapering abruptly to a thin neck, 13.80—46.4 x 0.42—5.13 um. Conidia hyaline, oval
or briolette, smooth-walled, 2.24—3.61 x 1.49-2.70 um.

Host. Larva of Elateridae (Coleoptera).

Habitat. The hosts were buried in soil, and the stroma were found in the leaf litter
on the forest floor.

Distribution. Vietnam.

Figure 3. Ophiocordyceps bidoupensis A—C fungus on an Elateridae larva D, E cross-section of the ascoma

showing the perithecial arrangement FH asci I ascospores J, K colony on PDA medium L=N conidiog-
enous cells and conidia O conidiogenous cells P, Q conidia. Scale bars: 1 cm (A=C); 200 um (D); 20 um
(E-H); 10 um (1); 2 cm J, K); 5 um (L-Q).

Multigene phylogeny and morphology of two new species 124

Notes. Phylogenetic analyses showed that O. bidoupensis was clustered with
O. houaynhangensis, O. brunneipunctata, O. langbianensis, O. cossidarum, and O. furca-
tosubulata of the O. sobolifera clade (Fig. 1). Their hosts were larvae of Elateridae com-
pared to cicada nymph hosts of the other species of the O. sobolifera clade (Table 2).
Ophiocordyceos bidoupensis was well-supported by bootstrap support and posterior
probabilities, and formed a separate subclade with O. houaynhangensis, O. brunnei-
punctata, O. langbianensis, and O. cossidarum. The morphology of O. bidoupensis was
clearly different in shape and size from other species of O. sobolifera clade (Table 2).
The stroma of O. bidoupensis grew solitary from the head of the host; sterile apices of
the stroma were different from the other species.

Discussion

Ophiocordyceps is the largest genus in the Ophiocordycipitaceae, with a wide range of
hosts and various species. At present, more than 290 species of Ophiocordyceps have
been reported (Index Fungorum 2022). However, only 11 species are described in the
O. sobolifera clade and their hosts are mainly Coleoptera larvae and cicada nymphs
(Hemiptera) (Table 2). We describe the new species O. hydrangea attacking cicada
nymphs and the new species O. bidoupensis attacking Coleoptera larvae. Most spe-
cies have diverse macro-morphological or micro-morphological characteristics due
to the same entomopathogenic fungi having a different host, or different species of
entomopathogenic fungi having the same host (Sung et al. 2007, 2011; Aratijo et
al. 2015; Araujo and Hughes 2016; Shrestha et al. 2016; Luangsa-ard et al. 2018;
Crous et al. 2019; Fan et al. 2021; Wang et al. 2021a). Hemiptera hosts are widely
present among the species of Ophiocordyceps, including species of the Hirsutella clade,
O. sobolifera clade, O. sphecocephala clade, and O. ravenelii clade.

The entomopathogenic fungi whose host is Hemiptera have diverse morphological
characteristics. For example, O. nutans (Patouillard) G.H. Sung, J.M. Sung, Hywel-Jones
& Spatafora (Sung et al. 2007), its hosts were stink bugs (Hemiptera), stromata solitary
or multiple, fertile parts was red (Hywel-Jones 1995a; Luangsa-ard et al. 2008), stromata
of O. brunneinigra (Hemipteran host) were flexuous, arising from between the head and
the thorax of the host (Luangsa-ard et al. 2018), stromata of O. spataforae Tasanathai,
Thanakipipattana, Khonsanit & Luangsa-ard were cylindrical, cream to pale brown
(Luangsa-ard et al. 2018). However, from previously reported Hemipteran hosts, only a
few hosts of the O. sobolifera clade were cicada nymphs in Ophiocordyceps (Kobayasi and
Shimizu 1963; Sung et al. 2011; Crous et al. 2019). In this study, the host of O. hydrangea
was a cicada nymph. More interestingly, the O. hydrangea was significantly more beauti-
ful than other species; the stroma grew from the head of the host cicada nymph, and the
top of the stroma like a hydrangea (Sung et al 2007, 2011; Crous et al. 2019). Coleoptera
hosts were common in species of Ophiocordyceps. More than 20 species of Ophiocordyceps
were parasitic on Coleoptera larvae (Shrestha et al. 2016). These species included O.
acicularis (Ravenel) Petch (Petch 1933), O. annulata (Kobayasi & Shimizu) Spatafora,
Kepler & C.A. Quandt (Kobayasi and Shimizu 1982; Spatafora et al. 2015), O. aphodii

109-130 (2022)

Weigiu Zou et al. / MycoKeys 92

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124 Weiqiu Zou et al. / MycoKeys 92: 109-130 (2022)

(Mathieson) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora (Mathieson 1949; Sung
et al. 2007), O. brunneipunctata (Hywel-Jones) G.H. Sung, J.M. Sung, Hywel-Jones &
Spatafora (Hywel-Jones 1995b; Sung et al. 2007; Luangsa-ard et al. 2008), O. furcatosub-
ulata H. Yu, Y. Wang & Y.B. Wang (Wang et al. 2021a), O. houaynhangensis Keochan-
pheng, Thanakitp., Mongkols. & Luangsa-ard (Crous et al. 2018), O. langbianensis'T.D.
Lao, TLA.H. Le & N.B. Truong (Lao et al. 2021), O. melolonthae (Tulasne & C. Tulasne)
G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora (Sung et al. 2007), and O. ravenelii
(Berkeley & M.A. Curtis) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora (Sung et al.
2007). Most species with Coleopteran host occur in soil and have solid, cylindrical, and
yellow stromata. This is consistent with the results of this study.

Phylogenetic analyses based on the data from five genes showed that our phylo-
genetic framework of Ophiocordyceps was consistent with previous studies (Sung et al.
2007, 2011; Quandt et al. 2014; Simmons et al. 2015; Crous et al. 2018, 2019; Wang
et al. 2018, 2021a; Lao et al. 2021). The genus of Ophiocordyceps consists of four clades,
including the Hirsutella clade, O. sobolifera clade, O. sphecocephala clade, and O. rav-
enelii clade. Phylogenetic analyses showed that O. hydrangea clustered with O. sobolif-
era, O. longissima, and O. yakusimensis in the O. sobolifera clade, and O. bidoupensis
clustered with O. houaynhangensis, O. brunneipunctata, O. langbianensis, O. cossidarum,
and O. furcatosubulata in the same clade. Species within the O. sobolifera clade had dif-
ferent hosts, and morphological characteristics. ‘These two new species clustered in two
separate subclades within the O. sobolifera clade. The hosts of one subclade were cicada
nymphs with stromata cylindrical or sarciniform, bright-colored, conidia were macro
(Kobayasi and Shimizu 1963; Crous et al. 2019), and the hosts of another subclade
were Coleoptera, with stromata cylindrical, conidia small, and a sterile apex on top of
the stroma (Hywel-Jones 1995b; Luangsa-ard et al. 2008; Crous et al. 2018; Lao et
al. 2021; Wang et al. 2021a). Therefore, the species of the O. sobolifera clade could be
divided into two separate subclades when more materials were collected.

The species of O. sobolifera clade had diverse morphological characteristics
(Table 2). The entomopathogenic fungi with cicada nymph hosts shared similar
characteristics, stromata solitary or multiple, cylindrical, and bright-colored. How-
ever, they also differed in morphology. For example, O. sobolifera lacked a protruding
ostiole with immersed perithecia (Kobayasi and Shimizu 1963), and this seems to be
contrary to O. yakusimensis (Kobayasi and Shimizu 1963). Stromata of O. longissima
were longer than other species, and had a short neck in perithecia (Sung et al. 2011).
Compared to the ovoid perithecia of O. longissima and O. yakusimensis, O. khonkae-
nensis was flask-shaped (Crous et al. 2019). The top of the stroma of O. hydrangea
was similar to hydrangea, the size and shape of conidiogenous cells and conidia were
different from O. khonkaenensis (Table 2). The entomopathogenic fungi using Coleop-
tera hosts shared similar characteristics, such as stromata solitary, cylindrical, sterile
apices on top, bright-colored. However, they had different shape and size of perithecia,
asci, ascospores, conidiogenous cells, and conidia. The perithecia of O. bidoupensis
was pyriform to lanceolate and brown-yellow. It was similar to O. brunneipunctata,
O. furcatosubulata, and O. langbianensis, and only O. houaynhangensis was clavate

Multigene phylogeny and morphology of two new species 125

(Hywel-Jones 1995b; Luangsa-ard et al. 2008; Crous et al. 2018; Lao et al. 2021;
Wang et al. 2021a). Conidiogenous cells of O. bidoupensis were cone-shaped, forming
on hyphae, with a hypertrophic base, tapering abruptly into a thin neck, smooth-
walled, with a smaller thin neck (0.42 um wide) than O. brunneipunctata (0.5 um),
O. furcatosubulata (0.9 um), and O. houaynhangensis (0.5 um).

Due to the unique geographical locations and climate conditions in China and
Vietnam, these areas contain a rich species diversity of Cordyceps s.l. However, our
survey of Cordyceps s.l. in China and Vietnam only represented a small portion of the
total. More samples of Cordyceps s.1. will continue to be collected in China and South-
east Asia in order to uncover additional undescribed taxa, and revise species with the
incorrect classification position of this group.

Acknowledgements

This work was funded by the National Natural Science Foundation of China
(31870017, 32060007).

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